PENAPISAN AKTIVITAS ANTIPROTOZOADALAM BIJI SAGA

(Adenanthera pavonina LINN)Lenny Sutedja

Puslitbang Kimia Terapan LlPI

INTISARIProtozoa Tetrahymena pyriformis GL adalah suatu eukaryot,

yang metabolismenya menyerupai mammalia, sehingga banyakdipergunakan sebagai mikroorganisma penguji dalam penentuankualitas protein juga dalam uji toksisitas. Dalam rangka pene-lusuran sifat toksis atau antinutrisi dalam biji saga (Adenan-thera pavonina LINN) sifat antiprotozoa dari ekstrak-ekstrak bijisaga yang diperoleh dari hasil ekstraksi berturut-turut dengan n-heksana dan etanol; telah diuji. Dari hasil pengamatan populasisel total dan sel hidup protozoa, didapatkan bahwa ekstrakminyak saga pada kadar sampai 0,1 % dalam medium per-tumbuhan tidak menunjukkan pengaruh yang nyata terhadappertumbuhan T. pyriformis GL selama 96 jam inkubasi pada30°C. Sedangkan ekstrak etanol saga menunjukkan hambatanyang nyata terhadap pertumbuhan T. pyriformis GL mulai 7 jaminkubasi pada 30°C. Kadar 0,1% ekstrak etanol saga dalammedium menghambat pertumbuhan T. pyriformis GL sebanyak55,1% dan 1% ekstrak etanol menunjukkan hambatan sebanyak87, 6% (pada 24 jam inkubasi). Ekstrak etanol saga menunjukkansifat antiprotozoa yang paling besar dibandingkan ekstraklainnya. Analisis kualitatif ekstrak etanol saga memberlkan hasilpositif terhadap saponin dan alkaloida dan hasil analisis dengankromatografi cair kinerja tinggi menunjukkan adanya palingsedikit delapan komponen dalam ekstrak etanol saga.

ABSTRACTThe protozoa Tetrahymena pyriformis GL is an eucaryote. Its

metabolism is similar to that of mammalia, so that it is widelyused as a biological tool in protein quality as well as toxicityassays. In the framework of searching toxic or antinutritiveproperties in saga seed (Adenanthera pavonina LINN), extractsof saga bean were tested for their antiprotozoa activity. The sagabean extracts were obtained after successive extraction with n-hexane and ethanol. Observation of total and motile cell popula-tion indicated that 0,1% saga oil in the medium did not showsignificant effect on the growth of T.pyriformis GL during 96hours incubation at 30°C. While ethanol extract of saga showedsignificant inhibition on the growth of T.pyriformis GL. Smallercell population was already observed at 7 hours incubation at30°C. At 24 hours incubation, 0,1% and 1% ethanol extract ofsaga in the medium showed 55,1% and 87,6% inhibitionrespectively. Ethanol extract showed the largest anttprotozoalactivity compared to the other extract. Qualitative analysisindicated the presence of saponin and alkaloid in the ethanolextract of saga bean. Chromatographic analysis with high

38

performance liquid chromatography showed the presence of atleast eight components in the ethanol extract of saga.

PENDAHULUANTetrahymena pyriformis termasuk phylum protozoa,

berbentuk lonjong dengan ukuran rata-rata 50x30f.l. Mudahtumbuh dalam perbenihan bebas bakteri atau pun dalamperbenihan sintetis. T.pyriformis merupakan eukaryot,organel-organel dan membran dinding sel sama dengan seleukaryot. Kebutuhan nutrisi dan metabolisma dalam tubuhT.pyriformis menyerupai metabolisma mamalia (hewantinggi), karena itu protozoa ini banyak dipergunakansebagai organisma penguji (1). Dibandingkan denganpenggunaan mammalia seperti tikus, uji toksisitas meng-gunakan protozoa dapat lebih cepat dan contoh yangdiperlukan lebih sedikit. Hasil penelitian Otsuka et al. (2)menunjukkan bahwa urutan daya toksisitas beberapa fungi-sida terhadap T.pyriformis adalah sama dengan terhadaptikus, sehingga toksisitas terhadap T.pyriformis dapat di-gunakan sebagai indeks toksisitas terhadap mammalia.

T.pyriformis telah digunakan sebagai organisma pengujidalam penelitian toksisitas a-tomatine (3), senyawa kimiadalam air limbah (4,5,6,7), fungisida (2), mikotoksin (8),insektisida (9), senyawa senyawa hasil fermentasi (10) danpenentuan aktivitas biologi rubratoksin A dan B (11).Penelitian-penelitian tersebut menggunakan T.pyriformisuntuk menguji toksisitas, dengan mengamati populasi sel,transmitans/absorbans atau kecepatan pemakaian oksigen(respiratory response). Mojzis et al (12) telah menelitipengaruh senyawa-senyawa dichlorvos dan polichlorinatedbiphenyls terhadap aktivitas enzim esterase, transferase,dehidrogenase dan fosfatase dalam Tetrahymena pyrifor-mis. Phillipson et al (13) melaporkan bahwa banyak tum-buh-tumbuhan tropis merupakan sumbersenyawa-senyawaantiprotozoa (13).

Dalam penelitian ini T.pyriformis digunakan sebagaimikroorganisma penguji dalam penapisan sifat anti protozoabiji saga. Seperti diketahui biji saga (Adenanthera pavoninaLINN) komposisi kimianya seperti kedeIai, mempunyaipotensi sebagai sumber protein nabati (14). Akan tetapipenelitian pendahuluan terhadap tikus menunjukkan bahwabiji saga mempunyai sifat antinutrisi, dimana didapatkan

JKTI, VOL. 5 - No.1, Juni, 1995

hambatan pertumbuhan tikus (15). Sehubungan dengan iniakan ditelusuri sifat toksis .atau antinutrisi biji saga, denganmenguji aktivitas ekstrak-ekstrak biji saga terhadapT.pyriformis.

BAHAN DAN METODABiji saga

Biji saga diperoleh dari Juana, Pati, Jawa Tengah.Organisma

Organisma yang dipergunakan ialah Tetrahymenapyriformis GL, yang berasal dari Macquarie University,Australia.Bahan kimia

Bahan-bahan kimia dengan mutu pro analisis dari E.Merck, bahan medium dari Difco, pelarut organik kualitasteknis yang didestilasi ulang, digunakan dalam penelitianini.

Pengolahan biji sagaBiji saga berkulit merah, keras dan tebal. Pemecahan

biji saga dilakukan dengan mesin penggiling sehinggaterbeIah menjadi dua bagian. Keping biji saga dipisahkandari kulit secara manual, kemudian dihaluskan sehinggadiperoleh tepung saga dengan ukuran 30/48 mesh.Ekstraksi biji saga

Keping biji saga diekstraksi dengan cara maserasi,berturut-turut dengan n-heksana dan etanol, seperti terlihatpada bagan 1. Pelarut diuapkan pada alat penguap berputarsehingga diperoleh ekstrak heksana (minyak saga; EH) danekstrak etanol saga (EEt) disamping residu heksana (RH)dan residu saga (RS).

BIJI SAGA

Ikulit saga63,26%

Ikeping biji saga

36,13%kadar air 14,3%

Idigiliug, dikeringkan

24 jam, 55'C, dibaluskan

tepung keping biji saga30148 mesh; leadar air 4,1%

dimaserasi dgn.n-heksana

residu beksan (RH) filtrat

dikeringkan 24 jam, 55'C, dibaluskan - 48180 mesbkadar air 3,9%dimaserasi dengan etanol

diuapkanvakum

minyak saga (Ell).26,97% darikeping biji saga

Ireside saga (RS) ekstrak biji saga

dalam etanol

I diuapkan vakum

ekstrak etanol saga (EEt)sirup kental coklat

10,17% dari keping biji saga

Bagan 1. Ekstr aksi keping biji saga dengan n-heksana dan etanol

JKTI, VOL. 5 - No.1, Juni, 1995

Kurva pertumbuhan T.pyriformis GL.Protozoa T.pyriformis GL ditumbuhkan dalam medium

yang terdiri atas 2% proteose pepton, 0,1% ekstrak ragi,0,1 % NaCl dan 0,5 % glukosa dengan pH 7,2 ; pada suhu29-30°C. Medium sebelumnya disterilkan dalam autoklafpada suhu 120°C, tekanan 1 kg/em- seIama 15 men it.Pengamatan dilakukan tiap empat jam, terhadap sel mati(nonmotile cells) dan sel total (setelah dinonaktipkandengan penambahan larutan Hayem) dengan mernper-gunakan hemasitometer Neubauer improved. Jumlah selhidup diperoleh dari hasil pengurangan sel total dengan selmati.

Uji aktivitas antiprotozoaMinyak saga (EH), ekstrak etanol (EEt), residu heksana

(RH) dan residu saga (RS) masing-masing ditambahkan kedalam 75 ml medium pertumbuhan protozoa dalam labuerlenmeyer, dalam variasi konsentrasi 0,001-1 %. Sebelum-nya RH dan RS dikeringkan pada 50°C seIama 24 jam,kemudian dihaluskan sampai lolos 80 mesh. Minyak sagadicampur dengan emulsifier Cremophore El (0,01 %) se-hingga diperoleh larutan homogen dengan medium. Steri-lisasi dilakukan pada suhu 120°C, tekanan 1 kg/cmt selama15 menit. T.pyriformis yang berumur 24 jam (57,2 x 104 selper rnI) sebanyak 3 ml diinokulasikan pada medium yangmerigandung ekstrak saga tersebut, kemudian perbenihandiinkubasi pada 30°C selama 96 jam. Pengamatan dilaku-kan terhadap jumlah sel mati dan sel total. Percobaanmenggunakan rancangan acak lengkap dengan masing-masing 3x2 ulangan. Data diolah dengan analisis variansipada taraf signifikan 5%. Persentase hambatan pertum-buhan dihitung dari rumus (K-S)/K x 100%, dimana K =jumlah sel hidup protozoa dalam medium tanpa ekstrak bijisaga (kontrol) dan S = jumlah seI hidup protozoa dalammedium yang mengandung ekstrak biji saga.

Pemeriksaan titokimia

Analisis kualitatif dilakukan terhadap ekstrak etanol(EEt) biji saga, dengan memeriksa adanya alkaloida(reagen Wagner, Dragendorf, Meyer), fenol (reagen diazoA & B), flavonoida (reagen etanol + NaOH 10%), saponin(tes kestabilan busa), tanin (reagen HCl), steroid/triterpen(reagen Liebermann Burchard) dan terpenoid (reagenvanilin + H2S04) (16).

Kromatograti cair klnerja tinggi (KCKl)Ekstrak etanol saga dianalisa dengan KCKT (Waters

associates) pada kolom RP18, eluen metanol:air = 8:2 (v/v)dengan kecepatan alir 0,7 ml/menit. Sensitivitas 0,02AUFS. Detektor yang digunakan ialah UV detektor padapanjang gelombang 313 nm. Sebe1umnya contoh yang akandianalisa dilarutkan dalam pelarut metanol:air=80:20 (v/v),dalam kadar 0,2% (b/v), kemudian disaring me1alui kertassaring Millipore, cat.no. FHLP 01300, filter type FH, poresize 0,5 um.

39

--' 105.€--'ur(/)

:I:«~

....J :::>E .<,....JUJ{J)

10'I

:5~103

~-,0 20 40 60 EjJ 100 120

WAKTU INKUBASI (JAM)

10°

HASIL DAN DISKUSIKurva Pertumbuhan

Kurva pertumbuhan T. pyriformis adalah seperti terlihatpada Gambar 1. Kecepatan pertumbuhan sangat pesatselama lebih kurang 24 jam terlihat dari kenaikan yangtinggi dari populasi seI protozoa (fasa logaritma). Setelahitu kecepatan pertumbuhan berkurang kemudian mulaistabil dan pada waktu inkubasi 56 jam mulai teramatiadanya sel protozoa yang mati. Sehingga setelah 56 jam,pengamatan dilakukan terhadap sel total dan sel hidup, dankurva pertumbuhan dibedakan antara kurva sel total dan selhidup (Gambar 1). Dapat dikatakan bahwa fasa stasionermulai teramati pada 56 jam waktu inkubasi. Sel protozoayang mati terlihat sebagai sel yang tidak bergerak danbentuk sel biasanya berubah dari lonjong menjadi bulat.

1~~--------------------------------,

o 10 20 30 I/j 50 eo 70 80 90 100 110

.WAKTU INKUBASI (JAM)

Gambar 1. Kurva pertumbuhan 'Lpyriformis GL (0) sel total, (D) sel hidup.

Pengaruh keping biji saga, residu heksan dan residusaga terhadap pertumbuhan protozoa

Pengaruh keping biji saga terhadap T.pyriformis, di-nyatakan dalam jumlah sel total dan sel hidup selamawaktu inkubasi seperti terlihat pada Gambar 2a dan 2b.Kadar 0,001 % dan 0,01 % keping biji saga dalam mediumselama 56 jam inkubasi tidak menunjukkan pengaruh yangnyata (P>0,05) terbadap populasi sel protozoa. Sedangkankadar 0,1 % dan 1 % keping biji saga menunjukkan ke-naikan populasi seI total T.pyriformis yang nyata (P<0,05)pada waktu inkubasi 24 dan 30 jam dibandingkan denganblanko. Setelah 30 jam, pengaruh keping biji saga terhadappopulasi sel total protozoa tidak nyata (P>O,OS, Gambar2b). Jadi jika diamati dari sel total saja, tak terlihat

40

pengaruh hambatan keping biji saga terhadap pertumbuhanT.pyriformis; tetapi yang teramati ialah kenaikan populasisel seperti yang disebutkan diatas. Akan tetapi sebenamyadalam perhitungan seI total, ikut terhitung juga sel yangmati, sehingga pengaruh biji saga akan lebih jelas jikaditinjau dari populasi· sel hidup. Seperti terlihat pad aGambar 2a, selama 30 jam inkubasi, populasi sel hidupprotozoa sarna dengan sel total, karena tidak teramatiadanya sel yang mati. Pada waktu inkubasi 48 jam mulaiterlihat adanya sel yang mati. Pengaruh 0,1-1 % keping bijisaga terhadap populasi sel hidup protozoa terlihat setelah48 jam, yang merupakan hambatan terhadap pertumbuhansel protozoa. Kadar 0,10 % menunjukkan hambatan nyatamulai 72 jam sedangkan 1 % menunjukkan hambatan nyatamulai 48 jam (P<0,05). Hambatan 1 % keping biji sagaberkisar antara 23,6% (48 jam) sampai 66,1 % (96 jam).

106 r------------------------------,Q

100o 20 .0 60 80

WAKTU INKUBASI (JAM)

120

Garnbar 2. Kurva pertumbuhan Tipyriformis GL dalam medium perturn-buhan yang mcngandung keping biji saga:

(a). pertumbuhan dinyatakan dengan perubahan jumlah sel hidup di dalarnmedium,

(b). pertumbuhan dinyatakan dengan pcrubahan jurnlah sel total di dalammedium.

(e) tanpa biji saga, (_) dengan biji saga 0,001 %, ("') dcngan biji saga0,01 %, (D) dengan bijisaga 0,1 % dan ( 0) dengan biji saga 1 %.

JKT1, VOL. 5 - No.1, Junl, 1995

EXPERIMENTAL PROCEDURESEXPERIMENTAL

I

In preparation for an experiment, the particle charge isaccurately weighed and placed in the empty drum. The airtable, nozzle, gas and slurry tubing are installed in theirposition using a clamp stand. The drum covers andsub covers are put in place. The wax for coating isaccurately weighed and placed in the feed wax beaker. Airflows to the nozzle and air table are turned on and set to thedesired level using the rotameters. The drive to the rollersis switched on and the drum speed set at the desired level.All thermometers and thermocouples are correctlyinstalled, then the heating plate and two air heaters areturned on. It takes approximately one hour to preheat theair and wax to the desired temperatures.

Once the air and wax have reached the desiredtemperature, the wax pump is switched on at a high rate topreheat the feed line without solidifying wax in the line.Once the molten wax has reached the spray nozzle, watchon, the flow rate is reduced to the desired level.

As particles begin to be coated by the wax, the air tableflow rate may need to be increased to ensure smooth flowof the particles across the air table. The base case operatingconditions for each particle size are particle charge of 750g, drum speed of 20 rpm, air nozzle flow rate 19.8 l/min,coating time is 15 minute. These conditions were used inall experiments, otherwise specified.

After the desired coating time, the experiment is shutdown. The slurry pump is switched off together with thestop watch to make sure the input feed time is defined. Thefluidising and atomising air are then switched off as well asall the electric powers. The stand clamp, covers, subcovers,air table and the nozzle are removed. The coated particlesare collected on the pan. Particles sticking to the drum wallare removed and weighed separately. The wax sticking tothe drum wall is scraped off as much as possible tocalculate the wax lost. The nozzle and the tube are weighedto calculate the amount of wax left in the feed lines. Therest of wax in the glass beaker was also weighed and thenet feed wax to the drum calculated by difference.

PARTICLE SIZE DISTRIBUTIONAt the completion of each experiment, the entire drum

contents was split into two parts. One part is used tomeasure the particle size distribution and fraction ofagglomerates formed. The other part was used to measurethe binder distribution and binder capture efficiency. Themass size distribution was measured using a set oflaboratory test sieves (BSS 410/1986). Particles wereplaced on the sieve stack (1 to 11.2 mm) and then put onthe shaker for 1 hour. Each fraction was weighed and themass fraction calculated.

The particle size distribution was expressed as thecumulative mass distribution F(x), which is the fraction ofthe total mass of particle that is less than size x, where xcan be expressed in terms of length [13].

AGGLOMERATE MASS FRACTIONAfter measurement of the particle size distribution by

sieving, the agglomerated particles were collected by handfrom each size fraction. The agglomerates were then

JKTI, VOL. 5 - No.1, Juni,1995

weighed to calculate the total mass fraction of agglomeratesformed. Note that sorting of agglomerates by hand wasonly necessary in size fractions in which both agglomeratesand coated single particles were found.

RESULTS AND DISCUSSIONPARTICLE GROWTH MECHANISM

In this experiment using model glass beads as particleand molten wax as binder it gives a simple growthmechanism, agglomeration or layering. When bindersprayed and hit the particle, the particle surface is wetted asa liquid layer. Before drying, the liquid layer build a liquidbridge to one another. If the binder is strong enough to holdparticles together, agglomerate resulted, if not, particlebreaking layering resulted.PARTICLE SIZE DISTRIBUTION

Examples of coated particle size distributions are shownin Figure 5. Smaller initial particle size gave a broaderparticle size distribution due to the higher proportion ofagglomerates formed. For larger initial particle sizes (:!: 3mm) the granule size distribution is much narrower. Thereare few agglomerates, and these are generally doublets ortriplets, rather than multi particle agglomerates. Clearly,prediction of the final particle size distribution is verydependent on knowing the extent of agglomerate formation.

120

100

80

60

40

20

00

~ 120

w 100>~ 80--' 60:::>~:::> 40uVl 20Vl-c~ 0

0120

100

80

60

40

20

00

Figure 5.

15mm

10 12

Feed 2mm

10 126 B

Feed 3 mm

2 6

SIEVE SIZE (mm)

8 10 12

Particle size distribution for different spray rates, particle sizesand amount of wax added.wax rate (g/min) Wax in (g)(e) 17,6 265(0) 31,6 315(_) 43,4 390(D) Initial size

21

AGGLOMERATE FORMATIONEffect of particle size

Figure 6 shows the mass fraction of agglomeratesformed as a function of initial particle size for 3 differentspray rates of molten wax. Clearly, particle size has asignificant effect on the formation of agglomerates. Forparticle sizes of 4 mm and above, there is virtually noagglomerate formation. As particle size is decreased below4 mm, there is sharp increase in the level of agglomerates.At 1.5 mm particle size, 60 to 90% of the original particlesare present as agglomerates, depending on spray rateconditions. These results match, at least qualitatively, thesharp transition from non-inertial to coating regimepredicted by Ennis [14].

U\llJI-<{0:lJJ:::<:o--'c»<!)<{u,o

zQI-U<{0:u,U\U\<{:::<:

FEE'D SIZE DIAMETER (mm)

Figure 6. Agglomerate fraction for different spray rates (yIax = 265 g).Wax rate (g/min):(e) 17.6(0) 31.6(\7) 43.4

Effect of wax spray rate and amount of binder addedThe effect of binder spray rate on agglomerates mass

fraction is also shown in Figure 6. Increasing the spray rateincreases the agglomerate formation in the criticaltransition (inertial) region but has no effect at large particlesizes (coating region). Alternatively, the effect of increasespray rate can be viewed as shifting to the right the curvesin Figure 6. Increasing the spray. rate from 17.6 to 43.4g/min increases the initial particle size at which 50%agglomerates occur from 1.7 to 2.8 mm.

Increasing the spray rate will increase the liquid layerthickness a particle picks up on each passage through thespray zone [15]. This increases the critical Stokes numberin the Ennis analysis and thus the probability of agglo-merate formation. In addition, the drying time for the liquidlayer will increase [15]. Particles will stay sticky for longer,increasing the chance of agglomerates forming.

22

In contrast, Figure 7 shows that agglomerate formationwas independent of the total amount of binder added at anygiven spray rate, within the experimental accuracy. Theamount of wax was varied between 35% and 52% of theinitial mass of particles in the drum. Clearly, the thicknessof the liquid layer at each pass through the spray zone,rather than the total (solid) coating thickness, is importantin determining agglomerate formation.

100 ,----------------,

~80

en~~ 60w~0...J~e-e 40u.0Z0i=0 20~u.enen« 0~

0 10FEED SIZE DIAMETER (mm)

Figure 7. The fraction agglomerates for different amount of wax addedat the same spray rate (43.4 g/min),Wax in (g):(0) 390(e) 265

Effect of drum speedRotation of the drum recycles of particles within the

drum and through the binder spray zone. Increase in speedincreases the mass of particles recycled per unit time. Asthe drum speed is raised from 20 to 40 rpm, the particlerecycle rate is estimated between 13.5 and 27 kg/min. Forthe same binder spray rate (g/min) the average amount ofbinder sticking on to the particles per pass through thespray zone should be proportional to drum speed. i.e. for adrum speed of 40 rpm the average layer thickness on theparticles should be half that at 20 rpm. Increase in speedshould reduce the average binder layer thickness [15],leading to growth by coating rather than coalescence.Increasing speed also increases the radial velocity of thedrum wall from 16.2 em/see at 20 rpm to 32.4 ern/see at40rpm. This may increase the particle relative velocity.

The experimental results show drum speed has effect onthe agglomerate formation. The agglomerate mass fractiondecreased slightly by increasing drum speed, as shown inFigure 8. However, the effect is. small when compared tochanging the spray rate. Doubling the drum speed reduces

JKTI, VOL. 5 - No.1, Juni, 1995

the initial size for 50% agglomerate formation only from2.1 mm to 1.8 mm.

~(/) 60w40:W 3.~<.!)<.!)<1: 1,0lJ..0zQ 4.I-

~lJ.. 20(/)(/) 5.<1:~

° 6.0 2 6FEED SIZE (mm)

Figure 8. The fraction of agglomerates for different drum speed (sprayrate of 31.4 g/min).Drum rotation:(0) 20 rpm

(.) 30 rpm

(\7) 40 rpm

CONCLUSION

Study of laboratory scale FDG has been done and theequipment run well. The experiments were performed tostudy the effect of 3 process variables on agglomerateformation. Agglomerate formation increases with decreas-ing particle size, increasing binder spray rate, and dec-reasing the drum speed.

Particle size between 3 and 4 mm is the transition fromagglomerating to layering process. The spreading ofparticle size distribution increased with decreasing particlefeed size due to the present of agglomerates. Smallerparticle gave broader size distribution than bigger.

ACKNOWLEDGEMENT

This work was carried out in the particle grouplaboratory, Department of Chemical Engineering, TheUniversity of Queensland, Australia, under supervisor DrJD Litster.

JKTI, VOL. 5 - No.1, Juni, 1995

REFERENCES

1. C.E.Capes, "Particle size Enlargement", Elsevier,Amsterdam, Netherlands (1980), chap. 3 - 7.

2. R.D.Young, and I.W.McCamy, "TVA DevelopmentWork and Experience with Pan Granulation of Fertili-zers", The Canadian J. Chern. Eng., vol.4S, Feb.(1967).J.D.Litster, M.Desai, and AAAdetayo, "Modelling ofFertilizer Granulation Using Population BalanceTechniques", 18th Australasian Chem.Engng.Conf.,CHEMECA '90, (1990).T.Robinson, and B.Waldie,"Dependency of Growth onGranule Size in a Spouted Bed Granulator", TransIChemE, vol.S7, 121-127 (1979).O.Uemaki and K.B.Mathur,"Granulation of AmmoniumSulfate Fertilizer in a Spouted bed", I&E. ProcessDesign Develop., 15(4), 504- (1976).B.Waldie, and T.Robinson, "Granulation in SpoutedBeds - Attrition and others Mechanisms", PowderTechnology, 27,163-169, (1980).

7. P.J.Weiss, and AMeisen, "Laboratory Studies onSulphur- Coating Urea by the Spouted Bed Process",Can.J. Chern. , vol 61, 440-447, June (1983).

8. P.G.Smith and AW.Nienow, "Particle GrowthMechanisms in Fluidised Bed Granulation-I The effectof process variables", Chem.Eng.Sci., vol. 38, No.8,1223-1231 (1~83).

9. AW.Nienow, "Fluidised Bed Granulation and Coating:Application to Materials, Agriculture and Biotechno-logy", Sixth International Symposium' on Agglomeration(Agglos (93), Nagoya, Japan (1993).

10. AR.Shirley, Jr., L.M.Nunnelly, and F.T.Carney, Jr.,"Melt Granulation of Urea by the Falling-CurtainProcess" Ind.Eng.Chem.Prod.Res.Dev., 21, 617-620(1982).

11. Anon, "Fluid Drum Granulation Update", NitrogenIssue No.196, March.April, British Sulphur Pub., 31Mount Pleasant, London, England (1992).

12. A.MJ.Seto, "A New Route for Granular AmmoniumNitrate", Proc.Fert.Soc. No.296, 31pp (1990).

13. AD. Randolph and M.ALarson, "Theory of ParticulateProsesses". 2nd.ed. Academic Press, Inc. San Diego,Ca, USA (1988), chap. 1-2.

14. BJ.Ennis, G.Tardos, and RPtctter, "A microlcvel-based characterization or granulation phenomena",Powder Technologv, 65, 257-272, (1991).

IS. R. Sarwono, "F/llidiscd Drum Granulation". M.Eng.Sci. Thesis, Queensland Llni, Australia (1994).

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